Genome Projects and Gene technologies Flashcards
what are the three methods for producing fragments of DNA ?
-using reverse transcriptase
-using restriction endonuclease enzymes
-using a gene machine
what is recombinant DNA?
DNA of two different species/two types of organisms that have been combined
How can you make DNA Fragments with reverse transcriptase?
-mRNA that was transcribed for the desired gene used
-mRNA acts as a template for production of single stranded stranded cDNA using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with DNA helicase
-double stranded DNA is formed on the template of cDNA using DNA polymerase
How can you make DNA fragments with restriction endonuclease enzymes and what are they ?
-restriction endonucleases are enzymes that can cut DNA at a specific base sequence
-the base sequence they recognise is called the recognition or restriction site
-the enzyme cuts DNA between two specific bases (cleavage site)
-this produces sticky ends
-which result in one strand of the DNA fragment being longer than the other strand
- make it easier to insert the desired gene into another organism’s DNA as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA
sticky ends are used in genetic engineering. Explain how
-joining two pieces of DNA
-by complementary base pairing
what are endonucleases?
enzymes produced by bacteria to cut up viral DNA as a defensive measure
Explain what sticky ends are and their importance in recombinant DNA technology.
-DNA is cleaved at the restriction site
-restriction endonucleases cuts out the DNA fragment
-sticky ends are staggered pieces of DNA that are complementary to each other
-complementary bases of the two sticky ends pair up
-DNA ligase joins sugar phosphate backbone
-allows us to bind DNA from one organism to another provided
-the same restriction endonucleases is used
How do we use a gene machine to produce DNA fragments?
-sequence of bases determined
-triplets worked out and fed into computer
-computer designs oligonucleotides which are assembled into the desired gene
-PCR replicates gene
-gene into plasmid using sticky ends
-gene check using sequencing techniques
positive evaluation of using the gene machine
-quick
-no introns=prokaryotes cant splice
-any sequence of nucleotides
-accurate
why are the same restriction endonucleases used to cut the DNA and the vector?
ensures the sticky ends of the gene are complementary to each other
what are the two ways of gene cloning ?
-in vivo- inside living organisms
-in vitro- outside living organisms
recall how a gene is inserted into a plasmid vector (in vivo 1.)
-sticky ends are complementary since same RE ends are used
-promoter and terminator regions added
-DNA ligase forms two phosphodiester bonds
State the steps of introducing DNA to host cells (in vivo 2.)
-host bacterial cells are placed into cold calcium chloride solution to make cell walls more permeable
-plasmids are added and mixture is heat shocked wich encourages the cells to take in the plasmid
State the steps in identifying transformed cells (in vivo 3.)
-replica plating (making copies of colonies on agar plate)
-grow in tetracycline
-marker gene can code for antibiotic resistance so only transformed cells that have the marker gene will survive and grow
-or marker genes can code for fluorescence so when the agar plate is placed under a UV light only transformed cells with fluoresce
What are the three stages of PCR
-denaturation
-annealing
-extension
Describe the process of PCR
-DENATURATION: double stranded DNA is heated to 95c which breaks hydrogen bonds
-ANNEALING:temperature is decreased to 55c so that primers can anneal to the ends of the single strands of DNA which provides a starting point for DNA polymerase to bind
-EXTENSION: temperature is increased to 72*c (optimum temperature for DNA polymerase to join nucleotides ). Then DNA polymerase extends the primers and forms template strands
what are the roles of primers in PCR?
enables replication to start by keeping strands separate
what is a vector?
-carrier of DNA
-into host cell
Explain why promoter and terminator regions are added to DNA fragments that are used to genetically modify organisms
Promoter regions=
● Allow transcription to start by allowing RNA polymerase to bind to DNA
● Can be selected to ensure gene expression happens only in specific cell types
Terminator gene=
● Ensure transcription stops at the end of a gene, by stopping RNA polymerase
recall how a gene is inserted into a plasmid vector
-sticky ends are complementary since same RE ends are used
-promoter and terminator regions added
-DNA ligase forms two phosphodiester bonds
Describe how host cells are transformed using vectors
● Plasmids enter cells (eg. following heat shock in a calcium ion solution)
● Viruses inject their DNA into cells which is then integrated into host DNA
Explain why marker genes are inserted into vectors
● To allow detection of genetically modified / transgenic cells / organisms
○ If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
○ If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
● As not all cells / organisms will take up the vector and be transformed
Suggest how recombinant DNA technology can be useful in medicine
● GM bacteria produce human proteins (eg. insulin for type 1 diabetes) → more ethically acceptable than using animal proteins and less likely to cause allergic reactions
● GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper
● Gene therapy
Suggest how recombinant DNA technology can be useful in agriculture
● GM crops resistant to herbicides → only weeds killed when crop sprayed with herbicide
● GM crops resistant to insect attack → reduce use of insecticide
● GM crops with added nutritional value (eg. Golden rice has a precursor of vitamin A)
● GM animals with increased growth hormone production
Suggest how recombinant DNA technology can be useful in industry
● GM bacteria produce enzymes used in industrial processes and food production
Describe what a DNA probe is
-short
-single stranded length of DNA
-label attached
what are the four main uses of DNA probes?
-genetic screening for diseases
-personalised medicine; choosing effective treatments
-genetic counselling
-genetic fingerprinting
How to make a DNA probe ?
-make DNA with complementary base sequence to desired allele
-copy using PCR
-attach marker
how are DNA probes used to locate specific alleles of genes?
-sequence for muted allele identified
-probe made with complementary bases
-probe is labelled (fluorescent or radioactive) then replicated
-DNA sample obtained
-DNA made single stranded by heating
-probe added; joins by complementary base pairings
-wash to remove unattached probes
can now identify using x-ray imaging or UV light
Recall the process of genetic fingerprinting
-DNA extracted from sample
-DNA cut/hydrolysed into segments using restriction endonucleases
-required core sequences intact
-DNA fragments separated using electrophoresis
-mixture put into wells on gel and electric current passed through
-immense gel in alkaline solution (hence two strands of DNA separated
-cover with nylon/ absorbent paper (to absorb DNA)
-radioactive marker/probe added/complementary to VNTRs
-areas with probe identified using x-ray film
explain the technique of gel elecrophoresis
-used to separate fragments of DNA in size order
-DNA fragments on agar gel with voltage across it
-negatively charged DNA moves towards the anode (positive)
-larger fragments move slower
-therefore less distance travelled in a fixed time
-separate strands using alkali-apply nylon membrane
-fragments labelled with DNA probes
-determine final position on gel by applying x ray film on the gel
-radioactivity from the DNA exposes the film and maps the fragment
what are the 4 steps of genetic fingerprinting
-EXTRACTION: extract DNA from sample
-DIGESTION: restriction endonucleases cut DNA into fragments
-SEPARATION: separate fragments using gel electrophoresis and transfer gel to nylon membrane
-HYBRIDISATION: add dNA probes to label DNA fragments
-DEVELOPMENT: nylon membrane with DNA fragments placed in x-ray
